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Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function.

ABSTRACT: Modulation of endothelial calcium-activated potassium (K_Ca) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of K_Ca channels with small (K_Ca2.x) and intermediate (K_Ca3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of K_Ca2.3 and large K_Ca conductance (K_Ca1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 μM) was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO) synthase, and blockers of K_Ca2.x and K_Ca1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 μM) markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP) increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of K_Ca2.x and K_Ca3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of K_Ca2.3 channels has a potential for treatment of erectile dysfunction.

SUBMITTER: Comerma-Steffensen SG

PROVIDER: S-EPMC5619997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and purposeControlling vascular tone involves K(+) efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K(Ca)2.3 and K(Ca)3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.Experimental approachTransgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K(Ca)2.3 and K(Ca)3.1 expression by immunohistochemistry and RT-PCR. K(Ca) currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K(Ca)3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.Key resultsK(Ca)3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K(Ca)2.3 and K(Ca)3.1 mRNA expression. With 200 nM [Ca(2+)](i) , the K(Ca)2.1-2.3/K(Ca)3.1 opener NS309 increased whole-cell currents. CyPPA, a K(Ca)2.2/K(Ca)2.3 opener, was without effect. With 1 µM [Ca(2+)](i) , the K(Ca)3.1 inhibitor TRAM-34 reduced currents whereas apamin (K(Ca)2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K(Ca)3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K(Ca)3.1 and the large-conductance K(Ca) channel, K(Ca)1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.Conclusion and implicationsAstrocytes express functional K(Ca)3.1 channels, and these contribute to neurovascular coupling.

Project description:ObjectiveTo investigate coronary endothelial protection of a small-conductance calcium-activated potassium (SK) channel activator against a period of cardioplegic-hypoxia and reoxygenation (CP-H/R) injury in mice and patients with diabetes (DM) and those without diabetes (nondiabetic [ND]).MethodsMouse small coronary arteries/heart endothelial cells (MHECs) and human coronary arterial endothelial cells (HCAECs) were dissected from the harvested hearts of mice (n = 16/group) and from discarded right atrial tissue samples of patients with DM and without DM (n = 8/group). The SK current density of MHECs was measured. The in vitro small arteries/arterioles, MHECs, and HCAECs were subjected to 60 minutes of CP hypoxia, followed by 60 minutes of oxygenation. Vessels were treated with or without the selective SK activator NS309 for 5 minutes before and during CP hypoxia.ResultsDM and/or CP-H/R significantly inhibited the total SK currents of MHECs and HCAECs and significantly diminished the mouse coronary relaxation response to NS309. Administration of NS309 immediately before and during CP hypoxia significantly improved the recovery of coronary endothelial function, as demonstrated by increased relaxation responses to adenosine 5'-diphosphate and substance P compared with those seen in controls (P < .05). This protective effect was more pronounced in vessels from ND mice and patients compared with DM mice and patients (P < .05). Cell surface membrane SK3 expression was significantly reduced after hypoxia, whereas cytosolic SK3 expression was greater than that of the sham control group (P < .05).ConclusionsApplication of NS309 immediately before and during CP hypoxia protects mouse and human coronary microvasculature against CP-H/R injury, but this effect is diminished in the diabetic coronary microvasculature. SK inhibition/inactivation and/or internalization/redistribution may contribute to CP-H/R-induced coronary endothelial and vascular relaxation dysfunction.

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Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function.

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